ALS-associated TDP-43 Aggregation: A Drosophila Model and A Role for Autophagy

描述(由申请人提供)：本提案的长期目标是了解肌萎缩侧索硬化症(ALS)背后的病理机制。最近，在散发性肌萎缩侧索硬化症患者的运动神经元包涵体中发现了TDP-43蛋白，并发现TDP-43基因突变与部分家族性ALS病例有关。这项拟议的研究试图确定TDP-43聚集在神经元中的后果，并确定潜在有毒的TDP-43聚集可以从受影响的细胞中清除的机制。这项建议的一个关键方面是建立TDP-43对黑腹果蝇的毒性模型。TDP-43将在果蝇的神经元中表达，并将分析与TDP-43毒性相关的表型。然后，包括无偏筛选在内的基因技术将被用来识别消除这些表型的因素。在这些检测中发现的基因可能会导致对疾病机制的新理解，或者成为未来治疗的目标。此外，Ubiqulin，一种最近发现的TDP-43相互作用蛋白，将使用该模型来探索其作用。泛素与未折叠的蛋白质反应密切相关，并被认为在神经元中具有保护功能。这一假说将直接在体内用果蝇进行验证。这项提案的另一个目标是确定自噬在TDP-43包涵体降解中的作用。自噬是细胞质成分的大量降解，包括错误折叠和聚集的蛋白质，对正常的神经元稳态至关重要。药理学、显微镜和生化技术将被用来探索自噬参与TDP-43聚集清除的假设。此外，上述果蝇模型也将在体内用于解决这一问题。 公共卫生相关性：如果自噬确实被发现对有毒的TDP-43聚集体的降解很重要，那么增强自噬的药物可能值得进一步探索作为潜在的治疗方法。重要的是，ALS及相关疾病的发病率、死亡率和社会经济代价都很高。由于这项拟议的研究，对疾病机制的新见解将对理解和潜在地治疗这种毁灭性的疾病至关重要。 注：下面以基本上未经编辑的形式提供了对个别审查员的批评。这些评论是在审查会议之前编写的，在会议讨论之后可能没有更新或修订。因此，它们可能不能完全反映个别审评员在小组讨论结束时的最后意见或小组的最终多数意见。以上讨论摘要概述了小组讨论的最终结果。

DESCRIPTION (provided by applicant): The long term objective of this proposal is to understand the pathologic mechanisms behind the disease amyotrophic lateral sclerosis (ALS). Recently, the protein TDP-43 was identified in inclusions in motor neurons of patients with sporadic ALS, and mutations in TDP-43 have been found to be responsible for a subset of familial cases of ALS. The proposed research seeks to identify the consequences of TDP-43 aggregation in neurons and identify mechanisms by which potentially toxic TDP-43 aggregates can be cleared from affected cells. A key aspect of this proposal is to develop a model of TDP-43 toxicity in Drosophila melanogaster. TDP-43 will be expressed in the neurons of flies, and phenotypes associated with TDP-43 toxicity will be analyzed. Genetic techniques, including unbiased screens, will then be used to identify factors that abrogate these phenotypes. Genes identified in these assays could lead to new understanding of disease mechanisms or become future targets of therapy. In addition, the role of Ubiquilin, a recently identified TDP-43-interacting protein, will be explored using this model. Ubiquilin is intimately involved in the unfolded protein response, and it is thought that this protein has a protective function in neurons. This hypothesis will be directly tested in vivo using Drosophila. Another goal of this proposal is to determine the role of autophagy in the degradation of TDP-43 inclusions. Autophagy is the bulk degradation of cytoplasmic components, including misfolded and aggregated proteins, and is critical for normal neuronal homeostasis. Pharmacologic, microscopic, and biochemical techniques will be used to explore the hypothesis that autophagy is involved in TDP-43 aggregate clearance. In addition, the Drosophila model described above will also be used to address this question in vivo. PUBLIC HEALTH RELEVANCE: If autophagy is indeed found to be important for the degradation of toxic TDP-43 aggregates, drugs that enhance autophagy may warrant further exploration as potential therapies. Importantly, ALS and related diseases have high morbidity, mortality, and socioeconomic cost. New insights into disease mechanisms as a result of the proposed research will be critical for understanding and potentially treating this devastating disease. NOTE: The critiques of individual reviewers are provided below in an essentially unedited form. These critiques were prepared prior to the review meeting and may not have been updated or revised subsequent to the discussion at the meeting. Therefore, they may not fully reflect the final opinions of the individual reviewers at the close of group discussion or the final majority opinion of the group. The Resume and Summary of Discussion above summarizes the final outcome of the group discussion.